P1590: THE IFI27/TFEB PATHWAY MEDIATES THE IMPAIRED AUTOPHAGIC DEGRADATION OF ITP BONE MARROW MACROPHAGES, RESULTING IN IMPAIRED MEGAKARYOCYTE MATURATION IN ITP
Authors: Jin Wu, Qiu Sha Huang, Zhuoyu An, Peng Zhao, Haixia Fu, Xiao-Hui Zhang
Journal: HemaSphere (2023)

Topic: 32. Platelet disorders Background: The pathogenesis of ITP is complex and primarily manifests as autoantibody-mediated platelet destruction and impaired platelet production. Autophagy is a highly conserved cellular process that eliminates damaged organelles and promotes the degradation of misfolded proteins. Appropriate autophagy exerts a protective effect, while excessive autophagy may lead to cell death and increased inflammation. The specific role of bone marrow macrophages (BM MΦ) in regulating megakaryopoiesis in ITP patients has not been elucidated. To investigate the role of ITP bone marrow macrophages in pathogenesis, this study found that the IFI27/TFEB pathway mediates the impaired autophagic degradation of ITP bone marrow macrophages, resulting in impaired megakaryocyte maturation. Aims: To explore the effect of macrophage autophagy on impaired megakaryocyte maturation in ITP. Methods: Our study involved 15 consecutive ITP cases and 15 healthy donors. qT-PCR for gene expression, Western blot for cellular protein expression, immunomagnetic bead sorting technique for cell isolation and purification, immunofluorescent chemical staining of biopsied tissues for analysis of tissue antigen distribution, enzyme-linked immunosorbent assay (ELISA) for cytokine expression, fluorescent probe for ROS, and mitochondrial membrane potential T cells were transfected with RFP-GFP-LC3 autophagy double-labelled adenovirus and examined by confocal microscopy for autophagic flux, transmission electron microscopy for the number of autophagosomes and autophagic lysosomes, lentiviral overexpression vector for gene function, and cell co-culture system for cell interactions. Results: The expression of LC3-II/LC3-I, Beclin1 and P62 were all increased in ITP-Mφ, and the number of autophagosomes increased while the number of autophagolysosomes decreased, indicating that the autophagic degradation of ITP-Mφ was blocked. Immunofluorescence showed decreased TFEB expression in the nucleus of ITP-Mφ, and WB detected decreased expression of CSTB, LAMP1, and LAMP2 in ITP-Mφ, suggesting ITP-Mφ lysosomal dysfunction. Treating ITP-Mφ with the autophagosome-lysosome fusion inhibitor bafilomycin A1 (Baf A1), the lysosomal acidification inhibitor chloroquine, the siRNA of TFEB, and the IFI27-shRNA, IFI27-cDNA, and TFEB-cDNA lentiviral vectors verified that the IFI17/TFEB pathway mediated lysosomal function in ITP-Mφ deficiency, resulting in blocked autophagosomal degradation and impaired autophagic flux. The co-culture of treated macrophages with megakaryocytes was found to ameliorate the lysosomal defect in ITP-Mφ as well as the impaired maturation of megakaryocytes. Summary/Conclusion: The abnormally high expression of IFI27 in ITP bone marrow macrophages mediates the IFI27-TFEB signalling pathway, leading to defective mitochondrial and lysosomal function in ITP bone marrow macrophages, resulting in impaired autophagosomal degradation and impaired autophagic flux. The functional defects in ITP-Mφ affect the growth and development of megakaryocytes and platelet production and are involved in the pathogenesis of ITP. Keywords: Macrophage, Idiopathic thombocytopenic purpura (ITP)